Lentilactobacillus buchneri domination during the fermentation of Japanese traditional fermented fish (funazushi)

Abstract Funazushi is a Japanese traditional fermented fish made with boiled rice without the addition of microbial starter cultures. Isolates from various commercial funazushi products, as identified by 16S rDNA sequences, suggested that Lentilactobacillus buchneri strains are major lactic acid bacteria. Based on an analysis of the putative CRISPR (clustered regularly interspaced short palindromic repeat) region, the genetic diversity of L. buchneri strains was examined. The data suggested that the diversity of L. buchneri strains depended on the factories at which funazushi was produced. An analysis of samples during fermentation indicated that the transition of microbes occurred, and L. buchneri was the dominant species. To determine the factors associated with domination, bacteriocin production and environmental stress tolerance, including NaCl and organic acid (lactate and acetate) tolerance, were evaluated. L. buchneri isolates did not produce bacteriocin. Although the isolates did not exhibit NaCl tolerance, they displayed higher lactate tolerance than other lactic acid bacteria isolated during funazushi fermentation. Based on reports that L. buchneri can convert lactate to acetate, the previous and present results suggested that lactate tolerance and lactate conversion in L. buchneri could explain its domination in funazushi. Our study presented a model for the domination mechanisms of specific microbes in fermented foods by spontaneous fermentation.


| INTRODUC TI ON
Funazushi is a unique fermented fish ( Figure 1) preparation that is considered as a precursor of modern sushi. For approximately 1500 years (Ito, 2018), it has been made in Shiga prefecture, which contains Lake Biwa, the largest lake in Japan. Crucian carp (funa) and boiled rice are used as ingredients and fermented for more than 6 months to complete the fermentation (Tsuda et al., 2012).
Fermentation is a complicated process. The typical funazushi production process is illustrated in Figure 1. Even in recent fermentation processes, starter microbial cultures are not commonly used.
Environmental stress tolerance, the utilization of nutrients such as sugar and nitrogen in a microbial species, and the presence of antimicrobial substances can help microbial species grow solely in fermented foods (domination regarding cell number) (Li et al., 2020;Satora et al., 2020;Zhang et al., 2015). Because funazushi contains 2%-5% (w/v) NaCl and organic acids including 1% (w/v) lactic acid derived from LAB metabolism (Tsuda et al., 2012), tolerance to NaCl and organic acid stress might be critical factors for domination. It is known that many LABs produce bacteriocins, which are antimicrobial peptides effective against related LAB species (Eguchi et al., 2001;Kawamoto et al., 2002). To gain insights into such characteristics of LAB isolates, we employed cultivation-based methodologies.
In this study, we analyzed major LABs in funazushi produced on the eastern side of Lake Biwa, a main funazushi production area, and the results suggested that Lentilactobacillus buchneri dominates in funazushi. The genetic diversity of L. buchneri strains was evaluated by amplification of the genes in the CRISPR (clustered regularly interspaced short palindromic repeat) locus. We also described the factors affecting the domination by L. buchneri.

| LAB species from funazushi products and intermediate samples collected in the Shiga area of Japan
First, we isolated LABs from funazushi products (nine samples) produced on the eastern side of Lake Biwa in Shiga prefecture, Japan and purchased from grocery stores. LABs were isolated from each sample. In our experimental conditions, LABs were detected if their cell numbers exceeded 1 × 10 2 cells per gram of sample. The isolated strains were identified according to 16S rDNA sequence homology.
The data revealed that all isolates belonged to L. buchneri (Table 1), suggesting that the major species in funazushi is L. buchneri.
To gain insights into the LAB species, intermediate samples, including boiled rice and fish, during funazushi fermentation were obtained from funazushi producers. LABs were isolated and identified F I G U R E 1 Scheme of the funazushi production process. The point of fermentation with boiled rice was defined as the starting time of fermentation ( Table 2). The data suggested that various LAB species existed in quantities exceeding 1 × 10 2 cells per gram of sample in the early stage. In the later stage, L. buchneri was detected as a major species.
Taken together, these results suggest that domination by L. buchneri occurred during fermentation.

| Genotyping of L. buchneri strains isolated from funazushi
To determine the genetic diversity of the L. buchneri isolates, we next performed genotyping analysis as described by Daughtry (Daughtry et al., 2018). cas9 or the type II-A repeat spacer at the CRISPR region These results suggest that a funazushi sample contains several genotypes of L. buchneri strains. It is speculated that L. buchneri strains have critical characteristics promoting their domination in funazushi.

| Bacteriocin production abilities of the L. buchneri strains
To determine the effects of antimicrobial activity on domination, the bacteriocin production abilities of the L. buchneri isolates were evaluated. In this assay, we employed Enterococcus mundtii NBRC 13712 because it was sensitive to class II bacteriocins (Eguchi et al., 2001). The bacteriocin production abilities of the L. buchneri strains presented in Tables 1 and 2 were evaluated. In our experimental conditions, growth inhibition by culture supernatant was not detected in any L. buchneri strain ( Figure 3). This result suggests that the L. buchneri strains from funazushi exhibited little or no bacteriocin production; therefore, it was unlikely that bacteriocins contributed to the microbial domination of L. buchneri during funazushi fermentation.

| Stress tolerance of the L. buchneri strains
To examine the environmental stress tolerance of L. buchneri, the environmental stress tolerance of the isolates from commercial funazushi products or fermenting intermediates of funazushi was evaluated. The growth rates of the isolates were compared to those of Levilactobacillus brevis KTL3 and Lactiplantibacillus plantarum KTL12 (Table 2), which were isolated from fermenting intermediates, under high concentrations of NaCl, lactic acid, and acetic acid ( Figure 4). In the presence of 0.5 M NaCl, the growth of the L. buchneri strains and the L. brevis strain was inhibited by 20%-30% and 48%, respectively ( Figure 4a). The L. plantarum strain exhibited almost no growth inhibition in the highly salted MRS (De Man, Rogosa, and Sharpe) medium, indicating that L. buchneri is not endowed with enhanced salt tolerance. The sensitivity of the L. buchneri strains to acetic acid was the same as that of L. plantarum (approximately 40% inhibition, Figure 4c). Conversely, it was apparent that the growth of the L. buchneri strains was maintained at more than 80% of the control level in the presence of 0.5% lactic acid (Figure 4b). The growth rates of L. brevis and L. plantarum in the presence of lactic acid were 57% and 64% versus the control, respectively. These results strongly suggest that tolerance to a high concentration (1% (w/v)) of lactic acid in funazushi among TA B L E 2 Microbial strains that appeared at high frequencies during the fermentation of funazushi other putative stressors contributes to the growth advantage of L. buchneri in the later stage of fermentation.

| DISCUSS ION
We analyzed LABs in funazushi produced without the addition of microbial starters. LAB species isolated from commercial products differed significantly from those isolated from intermediate samples.
This suggested that the domination by L. buchneri occurred after the transition of various LAB species. To gain insights into this phenomenon, we employed cultivation strategies and characterized the isolates from funazushi, although recent studies frequently employed culture-independent strategies for microbial community analysis (Marui et al., 2015;Yap et al., 2021).
Tsuda et al. reported that many LAB species were predominant in funazushi (Tsuda et al., 2012). In our study, L. buchneri strains were detected as the dominant species. We consider that this difference might depend on the locations of the production facilities and the maturation stages of the products. We also consider fu- Genotyping of the CRISPR locus in L. buchneri strains demonstrated that the strains isolated from funazushi have distinct differences, which were also observed in the length of the repeat spacer locus among strains. It was intriguing that the genotypes of strains differed both between strains and within single samples. These results indicate that in the final fermentation step of funazushi, L. buchneri consists of several strains rather than a single strain.
Taken together, the results suggest that L. buchneri strains may share unique characteristics, such as environmental stress tolerance, for domination in fermented foods.
To determine the factors associated with domination, we have evaluated bacteriocin production and environmental stress tolerance in L. buchneri isolates. Although L. buchneri was reported to produce F I G U R E 2 Polymerase chain reaction (PCR)-based detection of the clustered regularly interspaced short palindromic repeat-CRISPR-associated protein (CRISPR-Cas) elements in Lentilactobacillus buchneri strains. Amplification of (a) cas9 or (b) the hypervariable type II-A repeat spacer in the CRISPR array in 14 L. buchneri isolates by PCR. The strains are listed in Table 1 (RZ1-11: left side) and 2 (KTL7, 9, and 11: right side). A 0.1-10 kb DNA ladder is presented on the left side of both electrophoresis images

F I G U R E 3 Bacteriocin production abilities of the Lentilactobacillus buchneri
strains. An aliquot of 20 μl culture supernatants of the isolated L. buchneri strains were spotted on an MRS (De Man, Rogosa, and Sharpe) agar plate seeded with Enterococcus mundtii. The tested culture supernatant of the strain is indicated below each spotted zone. Upper panels; RZ1-6, middle panels; RZ7-10, AM1, and bottom panels; KTL7,9,11,PC1,and PC2. PC1 (Yildirim et al., 2002), our isolates did not produce the antibacterial substances. Several researchers reported that bacteriocins play significant roles in microbial domination in fermented foods (Janssen et al., 2018;Plessas et al., 2011). We therefore think that antimicrobial substances do not influence bacterial domination during fermentation of funazushi. Conversely, we found that L. buchneri strains have high stress tolerance to lactic acid. It is reported that LABs are damaged by lactate, one of their own metabolites. It is possible that the tolerance to the main metabolite produced by LABs helps L. buchneri become the dominant species during extremely long incubation (6 or more months in the preparation of funazushi). In addition, L. buchneri is known to produce acetic acid together with lactic acid under both aerobic and anaerobic conditions (Heinl et al., 2012;Heinl & Grabherr, 2017). This species can also metabolize lactic acid to acetic acid and 1,2

| Medium and growth conditions
The MRS agar medium (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was used for the growth and maintenance of LABs.

| Funazushi samples
Commercial funazushi products were purchased from grocery stores located on the eastern side of Lake Biwa in Shiga prefecture, Japan.
Fermenting samples were provided by a funazushi company in Hikone City, Shiga prefecture.

| Isolation of LAB
Samples (approximately 0.5 g) of a commercial funazushi or fermenting intermediates were separated into fish and boiled rice and

| Taxonomic identification
Taxonomic identification of the isolated strains was based on a partial sequence of 16S rDNA (primer pairs: 341F and 806R) using a previously described method (Kawamoto et al., 2002

| Bacteriocin assay
The spot-on-lawn method as described by van Reenen et al. was used to determine antimicrobial activity (van Reenen et al., 1998). Briefly, the isolated L. buchneri strains were cultured overnight in 5 ml MRS medium. An aliquot of 20 μl culture supernatants of the L. buchneri were spotted on an MRS agar plate seeded with overnight culture of the test bacterial strain (Enterococcus mundtii NBRC13712) at 0.25% (v/v). The culture supernatants of two LAB strains, a Latilactobacillus curvatus and a Lacticaseibacillus casei, were employed as positive controls. They were isolated from the local pickles in Japan and confirmed to produce clear growth-inhibitory zone in our previous examination. The growth-inhibitory zones around the culture supernatants of L. curvatus and the L. casei were approximately 8 mm and 7 mm in diameter, respectively.

| Environmental stress tolerance assay
The LAB cells with an identical optical density (OD, 0.5) were inoculated in 5 ml of MRS broth containing 0.5 M NaCl, 0.5% lactic acid, or 0.5% acetic acid and anaerobically incubated at 30°C. The OD at 600 nm (OD 600 ) was measured at 24 h. The ratio of OD 600 relative to that with no stressor (control) was indicated. The mean and SD of OD 600 were calculated from triplicate results.

| Statistical analysis
Values were expressed as the mean ± SD. Differences in means between groups were tested by the Tukey-Kramer test using the js-STAR program (http://www.kisnet.or.jp/nappa/ softw are/star8/ info/new.htm). A p-value of less than 0.05 was considered statistically significant.

ACK N OWLED G M ENTS
The authors are grateful to Kotaro Ikejima, also known as Happy Taro, for providing funazushi and fermented food samples. This study was supported in part by a grant to the Microbial Resource Center for Fermentation and Brewing from Ryukoku University.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.